Automatic apparatus for determining the percentage population of particulates in a medium

ABSTRACT

New and improved automatic method and apparatus for differentiating and determining the respective populations of specific particulates in a medium, for example, leukocytes in a whole blood sample comprises means for treating a number of phased quotient streams, each containing a portion of such blood sample, so as to stain either a specific type or a specific class of leukocytes, and passing each quotient stream through a corresponding viewing chamber. The number of stained leukocytes to the number of both stained and unstained leukocytes in each sample portion passing through the corresponding viewing chamber are counted concurrently by photometric means, the ratio of such numbers indicating the population of such specific leukocytes or such specific class of leukocytes in the corresponding sample portion. Preferably, the counting of both stained and unstained leukocytes in the sample portions in the different quotient streams are counted to a same predetermined reference, whereby the respective populations of the specific types or specific classes of leukocytes are directly related and recorded in correlated fashion, and any variations in either the flow rate or the volume of the sample portions in the different quotient streams are only incidental. Also, when particular leukocytes of a same quotient stream are stained and counted as a class, comparison-type logical operations determine the respective populations of the individual leukocytes in such class.

United States Patent 1 Groner et al.

[ AUTOMATIC APPARATUS FOR DETERMINING THE PERCENTAGE POPULATION OF PARTICULATES IN A MEDIUM [75] Inventors: Warren Groner, Whitestone; Jacob Kusnetz, Tarrytown; Alexander M.

Saunders, Bedford Village, all of N.Y.

[73] Assignee: Technicon Instruments Corporation,

Tarrytown, NY.

22 Filed: Oct. 30, 1970 211 Appl. No.: 85,353

[52] US. Cl... 356/39, 356/181, 250/218 [51] Int. Cl. G01n 33/16, G01j3/46 [58] Field of Search 356/39, 102-104,

' 356/208; 250/222 PC; 235/92 CP, 92 PC [5 6] References Cited UNITED STATES PATENTS 3,497,690 2/1970 Wheeless, Jr. 250/71 2,875,666 3/1959 Parker et al. 356/39 3,510,632 5/1970 Strandberg 235/92 CP 3,413,464 11/1968 Kamentsky 356/39 X 3,523,733 8/1970 Kling et al. 356/39 2,847,162 8/1958 Meyer 250/222 PC 3,439,267 4/1969 Coulter et al 356/39 X 3,503,684 3/1970 Preston, Jr. et al. 356/39 3,512,002 5/1970 Decker 250/222 PC 2,824,488 2/1958 Bridges et al. 356/186 X OTHER PUBLICATIONS A Photoelectronic Instrument for Counting and Sizing Aerosol Particles British Journal of Applied Physics, Suppl. Vol. 3, 1954 8138-8143 The Shape of Ground Petroleum Coke Particles, British Journal of Applied Physics, Suppl. Vol. 3 pp. 516-581. Cell Sizing: A Light Scattering Photometer For Rapid Volume Determination" 40 Review Sc. Instruments pp. 1029-1032 August 1969. Instrumentation for Automated Examination of Cellular Specimens 57 Proc. of the IEEE pp. 2007-2016.

Cell Volume Distribution Pattern Analysis 54 Amer. Journal Clin. Pathol. August 1970, J. L. Ladinsky.

[4511 June 19, 1973 Scanning Laser Microscope 223 Nature August 1969, Davidouits et al.

Rapid and Reliable Differential Counts on Dilute Leukocyte Suspension 76 Journal Lab. Clin. Med. 3 Sept. 1970, Oberjat et al. i

Lillie, R. D., l-Iistopathologic Technic & Practical Histochemistry, Blakiston -Div., McGraw-Hill, N.Y., 3rd Ed.

Hewlett-Packard Catalogue, 1967, pp. 494-498.

Primary ExaminerRonald L. Wibert Assistant Examiner-Conrad Clark Attorney-S. P. Tedesco and S. E. Rockwell [57] ABSTRACT New and improved automatic method and apparatus for differentiating and determining the respective populations of specific particulates in a medium, for example, leukocytes in a whole blood sample comprises means for treating a number of phased quotient streams, each containing a portion of such blood sample, so as to stain either a specific type or a specific class of leukocytes, and passing each quotient stream through a corresponding viewing chamber. The number of stained leukocytes to the number of both stained and unstained leukocytes in each sample portion passing through the corresponding viewing chamber are counted concurrently by photometric means, the ratio of such numbers indicating the population of such specific leukocytes or such specific class of leukocytes in the corresponding sample portion. Preferably, the counting of both stained and unstained leukocytes in the sample portions in the different quotient streams are counted to a same predetermined reference, whereby the respective populations of the specific types or specific classes of leukocytes are directly related and recorded in correlated fashion, and any variations in either the flow rate or the volume of the sample portions in the different quotient streams are only incidental. Also, when particular leukocytes ofa same quotient stream are stained and counted as a class, comparison-type logical operations determine the respective populations of the individual leukocytes in such class.

21 Claims, 4 Drawing Figures Unified States Patent 1 [111 3,740,143

Groner et a]. 11 June 19, 1973 229 TIMER E n: 27% i Q z 02 519 Patented June 19, 1973 5 Shuts-Shut 2 INVENTORS WARREN GRONER JACOB KUSNEIZ MEXANIIR HSAUNDERS ATTORNEY Isa 1M,

0000 O O ({OPO AUTOMATIC APPARATUS FOR DETERMINING THE PERCENTAGE POPULATION OF PARTICULATES IN A MEDIUM BACKGROUND OF THE INVENTION 1. Field of the Invention This invention relates to apparatus and method for differentiating the various types of leukocytes, or white cells, in whole blood samples and, more particularly, to the precise determination of the respective populations of the various types of leukocytes present in whole blood samples by automatic and continuous techniques.

2. Description of the Prior Art The information represented by the respective populations of various types of leukocytes, or white cells, in the human blood provides a sound basis for diagnostic processes. It has been appreciated that each of the various types of leukocytes serves a different and particular function within the human body, and that the respective populations of the various types of leukocytes provides valuable information regarding particular disease processes, either infectious, inflammatory, immune, or neoplastic.

Generally, three distinct types of formed bodies are present in the whole blood, that is, erythrocytes or red blood cells, leukocytes or white blood cells, and thrombocytes or platelets. Various techniques have been developed in the prior art for differentiating and counted the various types of leukocytes present in a whole blood sample. Generally, such differentiation and counting is performed manually by a skilled technician, and is effected by visually examining a dried stained smear of human blood which has been dried on a glass slide. Such techniques have been described, for example, in Laboratory Medicine-Hematology, (3rd ed.) by J. B. Miale (The C. V. Mosby Company, St. Louis, Mo., 1967). In the staining process, cytoplasm surrounding the nucleus of the leukocytes and the nucleus proper are differentially stained, so as to provide spectral and morphological information. Generally, leukocytes are divided into two groupings, which are identified as granulocytes and agranulocytes, respectively. The granulocytes are identifiable as having granules in the cytoplasm which are somewhat selectively sensitive to particular chemical stains. The agranulocytes are those leukocytes with a relatively clear cytoplasm, wherein comparatively few granules are found. Accordingly, agranulocytes are less well differentiated from one another by staining techniques but, however, agranulocytes can be differentiated by their morphological properties.

The prior art manual techniques for differentiating and counting leukocytes in a blood sample are overly cumbersome and tedious. Generally, a technician manually smears a very small volume of blood sample onto a clean glass slide. The smeared blood sample is allowed to dry, and is subsequently treated with appropriate staining chemicals and fixatives, e.g. Wrights stain. The product is then examined under a microscope, the types of leukocytes being individually differentiated and counted by the technician as they are brought within the viewing field. The technique is tedious, and subject to human error. Also, only a very small number of leukocytes are examined, usually not over 100, and the sub-classification or differentiation of the various types of leukocytes is based on this small count, whereby only a very crude approximation of their respective populations can be obtained. Recent advances made in the technology have essentially extended the visual technique by substituting an electronic interface, including appropriate scanning and measuring devices, between the viewing field of the microscope and a computer. These techniques can be described, generically, as pattern recognition techniques, whereby multiple parameters, or vectors, of the individual leukocytes brought within the viewing field are measured. The computer performs a series of calculations based on these multiple parameters to identify and classify the particular type of leukocyte. Notwithstanding its precision, accurate pattern recognition techniques are comparatively slow, since the stained blood sample is scanned in a particular pattern, either manually or mechanically, and re-focusing by the technician often necessary before a particular leukocyte can be measured accurately. Admittedly, re-focusing and scanning of the slide may be effected automatically, but such automation would be extremely expensive. Without such automation, however, the system has no particular advantages, since the counting rate is limited due to the scanning and re-focusing processes. For example, in present-day pattern recognition equipments, a skilled technician can process leukocytes at the rate of about ten cells per minute. Accordingly, the large count necessary to provide a sound statistical basis for a meaningful measurement of the respective populations of the various type of leukocytes in a blood sample is prohibitively expensive and very time consuming.

Also, in the prior art, the differentiation of leukocytes has been effected by passing a blood sample along an optical or electronic system while suspended in either an aqueous or non-aqueous liquid. However, when such systems have employed staining techniques, these techniques have not been selective with respect to the individual types of leukocytes and, therefore, such systems are operative only to provide a total count, rather than a true differentiation and sub-classification, of the various types of leukocytes, or alternatively have based a differentiation of the leukocytes only on their morphological properties.

Regardless of the techniques used in the prior art, an accurate differentiation of various types of leukocytes in a blood sample along with a sound statistical basis for determining their respective populations, was not possible, except by very elaborate and complicated equipments. This invention appreciates that one of the major shortcomings of the prior art has [been that the automated differentiation of the various: types of leukocytes in a blood sample has been based mainly on a single class of parameters of the cell, i.e., morphological or spectral information. Moreover, accurate differentiation has been made more complicated since the common practice had been to identify each of the different types of leukocytes in a blood sample concurrently and each cell in serial-like fashion, since a single volume of sample has been used, which tends: to increase significantly the possibility of error in determining the respective population of the various types of leukocytes.

OBJECTS OF THE INVENTION Therefore, an object of this invention is to provide for an improved method and apparatus for differentiating the various types of leukocytes present in a blood sample.

Another object of this invention is to provide an automated method and apparatus for the differentiation of leukocytes present in a whole-blood sample and, also, to effect a subclassification of such leukocytes.

Another object of this invention is to provide an automated method and apparatus for concurrently determining the respective populations of one or more types of leukocytes present in a blood sample.

Another object of this invention is to provide a method and apparatus for automatically screening a number of successive blood samples, so as to determine the respective populations of various types of leukocytes present in such samples.

Another object of this invention is to provide a method and apparatus for differentiating and identifying particular types of leukocytes in a blood sample by both spectral and morphological information and in avoidance of human intervention.

Another object of this invention is to provide an automated method and apparatus for determining, on a selective basis, the respective population of a particular type of leukocyte present in a blood sample.

Another object of the invention is to provide an automated method and apparatus by which one or more types of leukocytes present in a blood sample passing along a continuous-flow system can be differentiated and their respective populations determined, on a selective basis, and independently of the volume or flow rate of such blood sample along such system.

Another object of the invention is to differentiate and determine the population of a particulate in a liquid medium.

SUMMARY OF THE INVENTION In accordance with the preferred embodiment of this invention, a plurality of blood sample are introduced consecutively as a stream into a continuous-flow system, similar to the type described in the L. T. Skeggs et al. U.S. Pat. No. 3,421,432, issued on Mar. 22, 1966, which sample stream is divided into a plurality of quotient streams. In each quotient stream, the erythrocytes are hemolyzed and the leukocytes are fixed by introducing appropriate chemicals into the individual quotient streams on a continuous basis, along with staining chemicals selective with respect to a particular type or class of leukocytes, whereby only that particular type or class of leukocytes are stained in each quotient stream. The selective staining of a particular type or class of leukocytes in each quotient stream allows for a direct and very reliable enumeration of the stained cells by automated means. Each type or class of leukocytes can be identified by a single parameter, such that the need for sensing multiple parameters and relating such parameters to identify a particular type or class of leukocyte, such as in pattern recognition techniques, is avoided.

In accordance with one feature of this invention, hereinafter described as volume accounting, a same predetermined total number of leukocytes, both stained and unstained, are counted in each of the quotient streams, while concurrently identifying and counting the number of stained leukocytes within that predetermined total number or reference. The counting of the total number of stained and unstained leukocytes and, also, the number of stained leukocytes in the quotient streams is effected in parallel and in a phased relationship, whereby the results can be correlated with respect to a same blood sample. Accordingly, the volume as well as the flow rate of the sample in each of the quotient streams are incidental to and do not affect the ultimate percent count of the stained leukocytes.

As hereinafter described, a particular type or class of leukocytes in each quotient stream is selectively stained, such that the presence of each stained leukocyte can be sensed by conventional absorption techniques, and the counting of stained leukocytes in the individual quotient streams can be effected concurrently and in parallel fashion. Also, to relate the individual quotient streams, the number of leukocytes, both stained and unstained, are concurrently detected by conventional light scattering techniques and counted. With respect to each particular quotient stream, the scatter signals are counted independently and, during such counting, the absorption signals are counted concurrently to accumulate the count of the selectively stained leukocytes. However, when the predetermined number of scatter signals have been counted, further counting of the selectively stained leukocytes in the particular quotient stream is inhibited. Accordingly, the absorption signals, indicative of the selectively stained leukocytes in the particular quotient stream, are only counted during that time interval required for the counting of that predetermined number of leukocytes, both stained and unstained, passing through the viewing volume of a flow cell. However, completion of the counting operation with respect to one quotient stream does not inhibit the count operation with respect to the remaining quotient streams. When the counting operations with respect to each of the quotient streams have been completed, the ratio of the counts of the scatter and absorption signals, respectively, yields the percentage, or population, of the particular type or class of leukocytes in the blood sample, which can be recorded in correlated fashion. When a specific class of leukocytes is counted, particular types of leukocytes in such class can be differentiated by appropriate logical operations between the parallel counting channels or by pulse-height discrimination techniques within the corresponding channel, as described. Because of the volume accounting, the system is substantially insensitive to variations in the flow rate of the individual quotient streams and, also, of the corresponding volumes of blood sample contained in such quotient streams.

The scatter signal, according to other features of this invention, serves a multiple function. For example, since the scatter signal identifies the presence of a leukocyte, either stained or unstained in the viewing volume of the flow cell, each scatter signal identifies a valid sample period, and is used as a control signal to operate the channel logic. As such, a more reliable statistical base for determining the population of the leukocytes is achieved, since the scatter signal can be used to sample the absorption singal, whereby electrical noise is gated and reduced, as described.

DESCRIPTION OF THE DRAWINGS These and other objects and features of this invention will be made clear by the following detailed description taken in conjunction with the accompanying drawings wherein;

FIG. 1 is a plan showing the intended arrangement of FIGS. 1A, 1B and 1C;

FIG. 1A is a schematic diagram of a continuous-flow multiple-channel system wherein leukocytes in a whole blood sample are stained on a selective basis;

FIGS. 18 and 1C are block diagrams illustrating logical systems for counting the various types of leukocytes in the system shown in FIG. 1A within a particular reference and for determining the respective percentage populations of such leukocytes in the whole blood sample. As shown, the logical systems of FIG. 1B are operative with respect to the eosinophils, lymphocytes, and neutrophils, whereas the logical systems of FIG. 1C are operative with respect to the basophils and monocytes.

DETAILED DESCRIPTION OF THE INVENTION appropirate Referring now to the drawings, the analytical system comprises a sample device 1 for supplying individual blood samples in succession along a conduit 3. Sample supply device 1 comprises an indexible turntable 5 carrying a plurality of sample receptacles 7 arranged in a circular row. Turntable 5 is indexed intermittently by appropriate drive means, not shown, to position sample receptacles 7, in turn, at a takeoff position beneath an aspirating probe 9. Aspirating probe 9 is controlled by appropirate mechanism 11, for example, as described in U.S. Pat. No. 3,134,263 issued on May 26, 1964, to move into and out of a sample receptacle 7 positioned at the takeoff position during the dwell time of tumtable 5, whereby a portion of the sample is aspirated and passed along conduit 3 for subsequent treatment and analysis, as hereinafter described. Also, a wash reservoir 13 is positioned adjacent turntable 5, and probe 9 is controlled by mechanism 11 to be immersed in the wash reservoir during the indexing of the turntable. Conduit 3 is connected in flow communication with each of pump tubes 15, 17 and 19 in peristaltic pump 21, which are continuously occluded by pump rollers, symbolically indicated at 23, against a platen 25. Accordingly, the stream directed along conduit 3 comprises successive blood samples aspirated from adjacent sample receptacles 7 and separated from each other by a sequence of air, wash liquid, and air segments. Since conduit 3 is connected to pump tubes 15, 17 and 19 by branching member 27, a portion of each segment, whether gas or liquid, in the sample stream is divided into a number of quotient streams, which are directed into the various analytical channels for the selective staining of leukocytes, as hereinafter described.

According to the preferred embodiment, the quotient stream passing along pump tube 15 is treated for the staining and counting of neutrophils and eosinophils, as a class, in analytical channel I and, also, for the selective staining of eosinophils in analytical channel II. The quotient stream along pump tube 15 is initially mixed with a stream of an appropriate chemical for fixing the leukocytes, e.g., formalin percent in 0.2M sodium phosphate buffer of approximately pI-I7.0, which is aspirated from a source, not shown, along pump tube 29. The stream of fixative passing along pump tube 29 is passed through anice bath 31, e.g., at 4 c for approximately 5 minutes, and segmented by air passing along pump tube 33, and joined into the quotient stream prior to passing through mixing coil 35. The fixative stream is initially cooled, so as to delay the chemical reaction which fixes the leukocytes until a thorough mixing of the fixative and quotient streams has been completed in mixing coil 35. The quotient stream passing from mixing coil 35 through a heating coil 37, e.g., at 51 for approximately 3.5 minutes, to support and accelerate the fixing of leukocytes. The fixed quotient stream from heating coil 37 passes, in turn, through cooling coil 39, e.g., at 4 c for approximately 2 minutes, to inhibit further chemical reaction and allow for the introduction of an appropriate lysing agent, e.g., acetic acid 10 percent in H O. The acetic acid is aspirated from source, not shown, along pump tube 41, joined into the quotient stream downstream of ice bath 39. When the acetic acid .and quotient streams have been joined, the resulting stream passed through a heating bath 43, e.g., at 51 0 for 1.5 minutes, such as to inactivate any catalase which might interfere with the subsequent chemical reactions, e.g., the preferential utilization of hydrogen peroxide, and, also, to complete the lysing of the erothocytes, i.e., hemolysis, by the acetic acid present in the quotient stream. The fixed and lysed quotient stream passes from the heating bath 43 and is re-sampled along pump tubes 45 and 47, so as to be passed as distinct quotient streams into analytical channels AI and All, respectively. The quotient stream passed along pump tube 45 into analytical channel AI is initially mixed with an appropriate substrate, for example, comprising hydrogen peroxide and a solution of 4-chloro-l-naphthol, together with a solvent of 2.2'-dioxyethanol, bufiered at a mildly acidic pH, of approximately 4.5, hereinafter referred to as dark dye, which are aspirated and pumped along pump tube 49 and 51, respectively, from respective sources, not shown, and mixed thoroughly while passing through mixing coil 53. Preferably, to accelerate mixing, the dark dye passed along pump tube 51 is segmented by a stream of air pumped along pump tube 55. Preferably, the air along pump tube 55 which is injected into the dark dye stream forms discrete air bubbles in such stream, which occur at a frequency substantially greater than the frequency of the inter-sample segmentation in the quotient stream, so as to enter into the intra-sample segmentation of the individual blood segments. As understood in the prior art, intra-sample segmentation ensures thorough and complete mixing of the different liquids while passing through mixing coils and, also, that interior surface walls of the conduits along which a liquid stream is passed are thoroughly scrubbed to prevent contamination between the successive sample segments.

In analytical channel Al, the dark dye passing along pump tube 51 and appropriately segmented by air injected therein along pump tube 55 is joined with a stream of hydrogen peroxide (H 0 passing along pump tube 49, so as to define the substrate. The substrate stream is thoroughly mixed in mixing coil 53 and is reactive to selectively stain both eosinophils and neutrophils as, for example, described in the copending U.S. patent application of H. R. Ansley et Ser. No. 85,333 filed on even date herewith. After mixing in coil 53, the substrate is joined with the quotient stream passing along pump tube 45, now fixed and lysed, and thoroughly mixed while passing through mixing coil 57. The stream is then passed through heating bath 59, e.g., at 37 c, for approximately 1.5 minutes, so as to accelerate the staining and color the of he eosinophils and neutrophils. The color development referred to is representing by the chemical equation:

peroxidase 4-chlorol -naphthol+I-l O precipitate A substantially similar procedure is followed in analytical channel All, with respect to the selective staining of the eosinophils in the quotient stream passed along pump tube 47. As hereinafter described, the actual count of the eosinophils is used to (1) determine the particular percentage population of eosinophils in the particular blood sample and (2) as a substraction factor to determine their percentage population of neutrophils from the class information obtained by the counting of the eosinophils and neutrophils stained along analytical channel Al. In analytical channel All, the quotient stream passing along pump tube 47 is mixed with a substrate, for example, comprising hydrogen peroxide and a solution of 4-chlor-l-naphthol, together with a solvent of 2.2-dioxi ethanol, buffered to an acidic pH of approximately 3.5, hereinafter referred to as acid dye, passed along pump tubes 67 and 69, respectively. The stream of acid dye passed along pump tube 69 is segmented by air introduced along pump tube 71. Complete mixing of the streams of acid dye and hydrogen peroxide to form the substrate is ensured by passage through the mixing coil 73. The substrate passing from mixing coil 73 and quotient stream passing along pump tube 47 are joined and thoroughly mixed in mixing its 75 to effect the staining and color development of eosinophils in the blood segment on a selective basis. Color development follows the reaction in the above equation, except the pH is 3.5 room temperature is used. Subsequent to color development the refractive index of the red cell membranes is matched, e.g., by the addition of propylene glycol, which is passed along pump tube 77 and joined into the quotient stream and mixed therewith in mixing coil 79. The quotient stream passing from mixing oil 79 and containing the selectively stained eosinophils is directed along conduit 81 to its corresponding flow cell arrangement 83.

The remaining analytical channels All! and AlV of the system are particularly adapted to selectively stain basophils and monocytes, respectively. For example, and referring to analytical channel All], the quotient stream passed along pump tube 17 is joined with a stream containing appropriate staining and lysing agents, e.g., 0.1 percent Neutral Red and 1 percent Saponin, buffered to approximately a pH of 5.5, passed along pump tube 85 and which has been previously segmented by air introduced along pump tube 87. The resulting sample-dye stream is thoroughly mixed in mixing coil 89, wherein dissolution of the erythorocytes is effected and staining of the leukocytes is commenced. The quotient stream passing from mixing coil 89 is joined with a stream of formalin passed along pump tube 91 and subsequently passed along mixing coil 93, for fixing the leukocytes in quotient stream. Subsequently, the quotient stream passing from mixing coil 93 and containing selectively stained basophils is joined with a stream of propylene glycol passed along pump tube 95, for matching indices of refraction within the quotient stream proper and passed along a final mixing coil 97 and conduit 99 to its corresponding flow cell arrangement 101. The described chemistry for selective staining of basophils in the whole blood sample is commonly referred to as the Basophil Neutral-Red Method and has been described at pages 1 l26l 127 of the above-identified book by John B. Miale, MD.

The final leukocytes to be selectively stained are the monocytes, which is effected in analytical channel AlV, for example, as also described in the aboveidentified H. R. Ansley et al. patent application. As illustrated, the quotient stream passed along pump tube 19 is initially joined with a stream of fixative, e.g., 20 percent formalin buffered to approximate a pH of 6 with 0.1M phosphate buffer, passed along pump tube 103, which has been previously segmented by air introduced along pump tube 105. The quotient and fixative streams are joined within a cooling bath 107, e.g. at 4 c, so as to inhibit all chemical reactions between cells or plasma and the fixative until, at least, thorough mixing is completed with mixing coil 109, also located in cooling bath 107. The stream passing from cooling bath 107 is joined with a substrate, e.g., a-napthol-butyrate, passed along pump tube 111 and mixed in mixing coil 113, wherein the leukocytes in the quotient streams are fixed and the substrate is acted upon. The fixed quotient stream passing from mixing coil 113 is joined with a stream of coupling reagent, e.g., hexazonium pararosanilin, passed along pump tube 115 and mixed within mixing coil 1 17, so as to selectively stain and develop color in the monocytes in the successive blood segments. The color is developed according to the following equation:

li use in monocyt e a. a-naphthol butyrate p naphthol pH 6.1, room temp b. a-naphthol+hexazonium pararosanilin colored precipitate The quotient stream passing from mixing coil 117 and containing selectively stained monocytes is subsequently joined with a stream of acetic acid passed along pump tube 119 for lysing the erythrocytes. The lysing of erythrocytes is accelerated by passing the quotient stream through mixing coil 121. Subsequently, the quotient stream passing from mixing coil 121 and containing selectively stained monocytes is joined with a stream of propylene glycol passed along pump tube 123, so as to more closely match the indices of refraction of the liquid stream and membranes of lysed erythrocytes, and further mixed along mixing coil 125 before being passed along conduit 127 to the corresponding flow cell arrangement 129.

The flow cell assemblies 65, 83, 101 and 129 associated with analytical channels I, II, III and IV, respectively, are preferably of the type shown and described in the above-identified patent application of A. Elkind et al. Basically, in each flow cell assembly, the quotient stream passed from the respective analytical channel is entrained within a sheath stream of inert liquid and coaxially confined by the latter, whereby the diameter of the quotient stream can be very substantially reduced. Accordingly, the leukocytes present in quotient stream, when passing through the viewing chamber 151 of the flow cell assembly, are concentrated and caused to flow in a more tandem fashion. Also, the provision of the liquid sheath about the quotient stream avoids the possibility of blockage of the viewing chamber by artifacts and other particulate matter. Preferably, the quotient stream is confined to a diameter of approximately 0.003 inches (75 microns), whereby coincidence of leukocytes flowing through the viewing chamber 151 are very substantially reduced; also, the diameter of the sheath stream is in the order of 0.010 inches (250 microns) to allow for passage through the corresponding viewing chamber 151 of any artifacts or particulate matter.

In the drawings, the flow cell assemblies 65, 83, 101 and 129 and associated photometric apparatus are identical in structure, and the same references have been used to identify corresponding structures. Each flow cell assembly has associated therewith photometric equipment for detecting the passage of each stained leukocyte by absorption techniques, for example, to effect the counting of stained leukocytes passing along the viewing chamber 151, and, also, for detecting the passage of both stained and unstained leukocytes passing along viewing chamber 151 by scattering techniques. As hereinafter described, the scattered signals are employed to effect the volume accounting techniques, whereby the analytical results of each of the analytical channels are positively related and, also, to provide appropriate control signals for the logic arrangements hereinafter described.

Flow cell assemblies 65, 83, 101 and 129 each comprises a flow cell 153 as in flow communication with the corresponding conduit 63, 81, 99 and 127 along which the quotient stream, fully stained and lysed, is passed from analytical channels 1, II, III and 1V, respectively. Each conduit 63, 81, 99 and 127 includes a debubbling structure which vents the conduit to atmosphere, whereby all segmentizing air bubbles, both intraand intersample, are removed and only the remaining liquid constituents pass to the corresponding flow cell 153 along conduit 157. Subsequent to debubbling, the quotient stream along the conduit 157 comprises adjacent blood samples separated only by a wash liquid segment, the latter preventing contamination between successive samples flowing to and along the flow cell 153. As illustrated, each quotient stream is directed through a first tube inlet 157, whose outlet is disposed concentrically with respect to viewing chamber 151 of the flow cell. The sheath liquid for confining the quotient stream is provided from a pressurized-bottle source 159, which is maintained at a constant positive pressure by combined action of pump 161 and pressure regulator 163. The output of the source 159 is connected along a distributing conduit 165 to each of the flow cells 153 through an adjustable flow-control valve 167. The sheath liquid passing through each flowcontrol valve 167 is introduced into the corresponding flow cell 153 along a second tube inlet 169. Each tube inlet 169 is positioned to introduce the sheath liquid, so

as to concentrically confine the quotient stream as it is caused to flow through the viewing chamber 151, as described in the above-identified Elkind et al. patent.

Since the hydraulic system associated with each analytical channel AI, All, All! and AIV is opened by venting the quotient stream at the debubbler structure 155, the quotient stream and the sheath stream are pulled through the flow cell assembly 153 by a constant negative pressure maintained within liquid-waste bottle 171 by the combined action of vacuum pump 173 and vacuum regulator 175. As illustrated, the output of each flow cell 153 is connected to waste bottle 17] along a conduit 177. Each conduit 177 includes high flowresistance coils 179, which are temperature compensated in heating bath 181 to stabilize the flow rate through the flow cell assembly 153 against minor variations of flow resistance in the hydraulic system and ensure that a constant flow rate is maintained through the corresponding flow cell assemblies 153. The use of temperature-compensated, high flow-resistance coils in continuous-flow systems yas been more-fully described in the co-pending deJong patent application.

Accordingly, each of the quotient streams which have been treated in analytical channels Al, All, All] and AW are passed through the corresponding flow cell assemblies 153 in proper phase relationship. While apparatus for independently adjusting the relative phases of the quotient streams has not been illustrated, such apparatus is known and has been described for ex ample, in the M. H. Pelavin US. Pat. No. 3,512,163, issued on May 12, 1970. The leukocytes in the quotient streams passing along each viewing chamber 151 are detected by scattering and absorption techniques concurrently. Apparatus'for the detection of such leukocytes by scattering techniques and by absorption techniques have been indicated schematically, since such apparatuses are well known in the art and have been described in Measurement of Small Particles Using Light-Scattering. A Survey of the Current State of the Art by A. E. Martens, Ann. N. Y. Acad. Sci. 158; 690-702; in Rapid Multiple Mass Constituent Analy sis of Biological Cells by L. A. Kamentsky et al., Ann. N. Y. Acad. Sci. 157: 310-323; and in Absorption Cytophotometry: Comparative Methodology for Heterogeneous Objects, and the Two-Wavelength Method by M. L. Mendelsohn, which appears in Introduction to Quantitative Cytochemistry, edited by George L. Wied, Academic Press 1966, pp. 201-214. As shown, light from a source 183 is collimated by a condenser lens arrangement 185 and directed through an aperture 189 defined in the aperture plate 187, so as to illuminate the concentrically confined quotient stream passing along the viewing chamber 151. The actual viewing volume wherein leukocytes are detected is determined, essentially, by the diameter of the quotient stream passing along viewing chamber 151 and the dimensions of aperture 189. Preferably, the dimensions of aperture 189 in the direction of flow of the quotient stream should be minimal, but related to the size of the cells to be measured and the flow rate of the quotient stream so as not to occlude portions of the cell or band-limit the response of either the scattering or absorption apparatus; also, the dimensions of aperture 185 transverse to the direction of flow of the quotient stream should encompass the entire diameter of the quotient stream and allow for any variations or undulations in the flow pattern. The light beam passing through the viewing chamber 151, therefore, is intercepted by each of the individual leukocytes in the quotient stream, both stained and unstained, and passed through a beam splitter 189. One beam of light passing through beam splitter 189 passes through aperture 191 defined in aperture mask 193 and is incident on a photodiode 195, for measuring the absorption of the light beam by each stained leukocyte. The second beam of light passing through beam splitter 189 is directed toward photomultiplier tube 197, which is discriminated by an aperture mask 199 having an annular aperture 201, such that only portions of the light beam scattered by the stained or unstained leukocytes in the quotient stream is incident on photomultiplier 197.

The absorption and scattering signals generated by the photodiode 195 and photomultiplier 197, respectively, associated with each of the analytical channels I, II, III, and IV are directed into corresponding logic channels Ll, LII, LIII, and LIV, whereby the respective populations of the leukocytes in the various quotient streams can be automatically determined and correlated with respect to a same blood sample.

To facilitate an understanding of the logical operations for particularly determining the respective popu lations of leukocytes, reference is initially made to logic channels LII and LIV, associated with analytical channels All and AIV and adapted to count the populations;

of eosinophils and monocytes, respectively. A same basic logic arrangement is included in each of logic channels LI and LIII, associated with analytical channels AI and AIII, respectively, along with additional logic units. As hereinafter described, logic channel LI is operative to determine the population of neutrophils, which have been stained in a class including eosinophils and the population of lymphocytes in the blood sample, as hereinafter described. In the interest of clarification, corresponding units in each of the logic channels LI through LIV have been identified by a same reference.

Referring to the logic channels LII and LIV, associated with the analytical channels AH and AIV, the scatter signals generated by photomultiplier 197 upon passage of either stained or unstained leukocytes through the viewing chamber 151 and the absorption signals generated by photodiode 195 upon passage of stained leukocytes through viewing chamber 151 are amplified by amplifiers 201 and 203, respectively. The outputs of amplifiers 201 and 203 are connected to the inputs of the comparator circuits 205 and 207 respectively, which are operated as thesholding devices, so as to effectively block any noise present on the amplified scatter and absorption signals, respectively. The output of the comparators 205 and 207 are connected, in turn, to the inputs of monostable multivibrators 209 and 211, respectively. The output pulses generated by multivibrators 209 and 211, respectively, correspond in time to the presence of a stained leukocyte, in the viewing chamber 151 of the corresponding flow cell 153; however, an output pulse generated only by multivibrator 209 corresponds in time to the presence of both a stained and an unstained leukocyte, in the viewing chamber 151 of the flow cell 153.

As shown, the output of multivibrator 211 is connected to one input of AND gate 213, whereas the output of multivibrator 209 is connected to the remaining input of AND gate 213 and to one input of AND gate 215, the output of AND gate 213 is connected to one input of AND gate 217. The remaining inputs of both AND gates 215 and 217 are connected to the set output of flip-flop 219. As hereinafter described, flip-flop 219 is set only when a previous counting operation has been completed and recorded by the printer 221 and a next counting operation is to be initiated. To this end, the set input of flip-flop 219 is connected to the output of AND gate 223. One input of AND. gate 223 is connected to printer 221 along lead 225. At the completion of each printing operation, as hereinafter described, printer 221 energizes lead 225. The remaining input of AND gate 223 is connected along lead 227 to the output of adjustable time device 229. Timer 229 is responsive to each indexing of turntable 5 to provide a control pulse along lead 227 which is delayed to coincide with the passage of the treated quotient streams of the blood sample being aspirated through the viewing chambers 151, respectively, associated with the various analytical channels. In operation, timer device 229 provides a series of control pulses which are in phase with the passage of the successive blood samples in the flow cell assemblies 153.

Assuming that the results of a previous counting operation has been printed by printer 221 and lead 225 has been energized, so as to condition AND gate 223,

a pulse provided by timer device 229 along lead 227 energizes AND gate 223 which, in turn, sets flip-flop 219. When flip-flop 219 has been set, each of the AND gates 21S and 217 is energized. The outputs of AND gates 215 and 217 are connected to the inputs of preset counter 231 and accumulating counter 233, respectively, which have been previously reset to zero by a reset pulse directed along lead 235 by printer 221 at the completion of a previous printing operation. As hereinafter described, AND gates 215 and 217 control the operations of preset counter 231 and accumulating counter 233, respectively, such that the respective populations of stained leukocytes stained in each of the quotient streams and present in a same original blood sample can be properly related and directly recorded.

Accordingly, the scatter pulses generated by multivibrator 209 in the basic logic arrangement pass through AND gate 215 to preset counter 231, whose count is indicative of the total number of leukocytes, both stained and unstained, passed through viewing chamber 151 of the associated flow cell 153 since the commencement of a counting operation, i.e., the setting of flip-flop 223. Concurrently, the absorption pulses generated by multivibrator 211 in the basic logic arrangement pass through AND gate 213 and AND gate 217 to the accumulating counter 233, whose count is indicative of the total number of stained leukocytes within the total count indicated by preset counter 231. Accordingly, the percentage population of the stained leukocytes being counted in any logic channel is given immediately by the ratio of the respective counts of preset counter 231 and accumulating counter 233. It can be appreciated that the larger the count provided for on the preset counter 231, the more accurate, on a statistical basis, is the determination of the population of the particular leukocyte. I

Due to the operation of AND gate 213, accumulating counter 233 is operative to count an absorption pulse only where coincident with a scatter pulse. In effect, AND gate 213 serves to sample the absorption signal, so as to substantially reduce the possibility of false" counts due to random large noise signals appearing at the output of absorption amplifier 203 and resulting, for example, from the passage of artifacts through the associated viewing chamber 151 or noise developed within the amplifier passing to multivibrator 211, and having a magnitude in excess of the threshhold in comparator 207. However, the occurrence of a large noise signal in the absorption channel concurrently with the generation of a scatter pulse by the multivibrator 209, due to the presence of an unstained leukocyte in the viewing chamber 151, can be passed through AND gates 213 and 217 and counted by accumulation counter 233.0n a statistical basis, this is very remote possibility, and the ratio of the respective counts in accumulating counter 233 and preset counter 231 provides a very reliable indication of the percentage population of the selectively stained leukocytes in the corresponding analytical channel. Preferably, preset counter 231 is limited at a predetermined count, for example, 10,000 or any other multiple of 100, whereas accumulating counter 233 is not so limited. When preset counter 231 has completed its count, for example, to 10,000, it inhibits further counting by the corresponding accumulating counter 233, and is operative to supply a control pulse along lead 237 to the reset input of flip-flop 219. Resetting of flip-flop 219 operates to inhibit AND gates 215 and 217 and to prevent any further counting of scatter and absorption pulses by preset counter 231 and accumulating counter 233, respectively. The output of preset counter 231 is also connected along lead 237 to a corresponding input of the start-scan AND gate 239, which initiates the operation of multiplexer unit 241, as hereinafter described. Each of the remaining inputs of start-scan AND gate 239 are connected to appropriate units in corresponding logic channels, wherein an indication is provided that all logic and counting operations in that particular channel have been completed and that information is available for the printer 221. Start-scan AND gate 239, therefore, operates to delay recording of information accumulated in each of logic channels LI, LII, LIlI and LIV, until the logic and counting operations in all such channels have been completed with respect to a particular blood sample, whereby such information can be correlated and recorded with respect to the particular blood sample and, hence, with the corresponding source individual.

If desired, the machine function of each logic channel can be monitored by a rate monitor 243 and a stylus recorder 245. The input of rate monitor 243 is connected to the output of multivibrator 209, and the output of rate monitor 243 connected to stylus-drive mechanism of stylus recorder 245. Rate monitor 243 operates to integrate scatter pulses generated by multi vibrator 209, and controls the deflection of the stylus. The deflection of the stylus in recorder 245 provides a continuous indication of the concentration of leukocytes passing through viewing chamber 151 of flow cell 153. The leukocyte concentration in each viewing chamber 151 varies due to the presence of a wash liquid segment between successive blood samples in the quotient stream, since air segments have been debubbled and, also, due to some intermixing between the wash liquid segment and the contiguous blood samples. While a blood sample of constant dilution is passing through the viewing chamber 151 of the corresponding flow cell arrangement 153, the stylus deflection, or trace, is essentially flat, so as to indicate a steady-state condition. Also, the control pulse generated by the timer device 229 is connected to rate monitor 243 along lead 227 and loads the rate monitor, so as to superimpose a blip on the trace, provided on the stylus recorder which indicates the commencement of a counting operation, and concurrently sets flip-flop 223, whereupon the scatter and absorption pulses are passed to preset and accumulating counters 231 and 233 respectively. If such blip does not coincide with the plateau of the trace, it is indicative that the counting operation is not properly phased with respect to the passage of blood sample in the quotient stream through the corresponding analytical channel and through the corresponding flow cell arrangement 153. In such event, the phasing, or running time, of the quotient streams to the corresponding flow cells 153 can be adjusted individually by structures known in the art and, for example, described in the above-identified M. H. Pelavin U.S. patent.

The logic operations hereinabove described are applicable where a single leukocyte is stained in a particular analytical channel and its respective percentage population is to be determined. The logic channels LII and LIV are adapted to determine the respective percentage populations of eosinophils and monocytes, respectively, within the total counts of the corresponding preset counters 231. When the counting operation in each logic channel has been completed, the particular count of the stained leukocytes remains in the corresponding accumulating counter 233, and is available for recording. Each logic channel is inhibited by the action of the corresponding preset counter 231 in resetting flip-fiop 219, whereby the blocking AND gates 215 and 217 are disabled.

The same basic logic arrangement, as described, is included in each of logic channels AI and AIII. In logic channel AI, however, additional logic circuitry is provided (1) for selectively discriminating the neutrophils from the total count of neutrophils and eosinophils, which have been selectively stained as a class in analytical channel LI and counted as a class in the corresponding accumulating register 233 and (2) for counting lymphocytes, which have not been stained in the analytical channel LI, by their size as manifested by scatter pulse-height discrimination. In logic channel III, additional logic circuitry is provided to selectively and positively spectrally discriminate the basophils, which have been selectively stained in analytical channel III, from noise signals, which have no particular spectral signature. In each of logic channel LI and LIII, those units included in the basic logic arrangement, as described, are identified by a same reference number.

Referring to logic arrangement LI, and in accordance with the above described, the absorption pulses are generated by multivibrator 211 upon passage of either an eosinophil or a neutrophil along the viewing chamber 151 of corresponding analytical channel AI. Accordingly, the count in accumulating counter 233 identifies the total population of these leukocytes within the count of preset counter 231 of logic arrangement LI. As described above, the count in accumulating counter 233 of logic channel LII identified the total count of eosinophils within the count of the corresponding preset register 231 in the segment of the same blood sample passed along analytical channel LII. The preset counters 231 in logic channels LI and LII are limited at a same count. As shown, the respective outputs of the accumulation registers 233 in logic channels LI and LII are connected to inputs of a subtract register 247. Subtract register 247 is connected to the output of AND gate 249, whose inputs are connected to leads 237 from preset counters 231 in logic channels LI and LII, respectively. Accordingly, when a counting operation has been completed in each of logic channels LI and LII,

AND gate 249 is enabled to trigger subtract register 247. Subtract register 247 operates to subtract the eosinophil count in accumulating counter 233 of logic channel LII from the total eosinophil-neutrophil count in accumulating counter 233 of logic channel LI, the difference being stored and indicating the total neutrophil count in the particular blood sample. When the subtract operation is completed, subtract register 247 energizes one input of AND gate 251, whose other input has been previously energized by preset counter 231 in logic channel Ll along lead 253. Energization of AND gate 251 indicates that logic arrangements LI and LII and subtract register 247 have completed their respective operations. AND gate 251, in turn, energies a corresponding input of start-scan AND gate 239. Since preset counters 231 in logic arrangements LI and LII, respectively, have been limited to a same count, the total eosinophil count in accumulation counter 233 of logic channel LII and the tOtal neutrophil count in subtract register 247 are properly related with respect to the same blood sample.

Additionally, logic arrangement LI includes additional logic to determine the percentage population of lymphocytes in the blood sample by pulse-height discrimination techniques. As lymphocytes and monocytes are agranulocytes, they are not stained, as are the eosinophils and neutrophils, in analytical channel AI. However, lymphocytes are readily discriminated with respect to monocytes by the marked differences in the respective sizes of their cellular structures. Normally, a lymphocyte has a much smaller diameter, e.g. up to approximately 7 microns, than the diameter of a monocyte which ranges from 12 and microns. Since the quantity of light scattered by a cellular structure is an increasing function of size, the presence of a lymphocyte in the viewing channel 151 of analytical channel LI can be detected and identified. To this end, the output of scatter amplifier 201 in logic arrangement LI is coupled to a second comparator 255 along lead 257. Comparator 255 provides an output only when the amplified scatter signal exceeds that magnitude generated by a monocyte passing through viewing chamber 151. The output of comparator 255 is connected along lead 259 to a multivibrator 261, whose output is connected to one input S H of anti-coincidence gate 263. The outputs of scatter multivibrator 209 and absorption multivibrator 211 in logic arrangement LI are connected to second and third inputs S and A, respectively, of anticoincidence gate 263. Since pulses applied at inputs 8, S and A of anti-coincidence gate 263 are generated in response to the presence of the same leukocyte in viewing chamber 151 of analytical channel AI, such pulses, if generated, are coincident. Anti-coincident gate 263 is operative to generate the function S 'S -A. That is, the absence of a signal at input A identifies the particular leukocyte in viewing chamber 151 as not being stained and as being an agranulocyte; the presence of a signal at input 8,, indicates that a leukocyte present in the viewing chamber has a size in excess of the normal size of a lymphocyte and, hence, a monocyte; and the presence of a signal at input 8,, indicates the presence of a leukocyte within the viewing chamber. Accordingly, anti-coincidence gate 263 discriminates the passage of a lymphocyte through viewing chamber 151 of analytical channel AI, according to the above function.

The output of anti-coincidence gate 263 is connected to one input of a second blocking AND gate 265, whose remaining input is connected to the set output of flip-flop 219 a logic channel L1. The output of blocking AND gate 265 is connected to the input ofa second accumulating counter 267, whose count is indicative of the number of lymphocytes included within the total count indicated by preset counter 231 in logic arrangement LI. The output of the accumulating register 267 is connected to a corresponding terminal of multiplexer 241. The operation of accumulating counter 267 is similar to that of accumulating counter 233 in logic arrangement LI, in that its operation is inhibited'upon resetting of flip-flop 219 by printer 221, as described, and it is reset by a control pulse passed along lead 235 by printer 221 upon completion of a print-out operation. Also, since the count in the accumulating register 267 is related to preset counter 231 of logic channel Ll, the operation of AND gate 251 to energize the corresponding input of start-scan AND gate 239 indicates the availability of the lymphocyte count.

With respect to the counting of basophils, which generally have a population less than 1 percent of the total leukocyte population in whole blood, logic arrangement LIII, as illustrated, is operative to discriminate any artifact, e.g., dust particle, gas bubble, etc., passing through the corresponding viewing chamber 151 and, also, random electrical noise which could produce a false count and materially affect the determination of the percentage population of basophils in the blood sample. To this end, a dichroic mirror is associated with beam splitter 189 in flow cell assembly 101, so as to reflect the blue component of the absorption signal to a photodiode 271 and to pass the red component to a photodiode 273. Each of the photodiodes 271 and 273 is shielded by an aperture plate 193, as described. In effect, logic channel LIII includes parallel absorption channels for processing the red and blue components, respectively, of the absorption signal. Since the basophils are stained red, they are absorbing of blue light. Accordingly, a stained basophil in viewing chamber 151 of analytical channel AIII, substantially reduces the intensity of light incident on photodiode 271; however, the presence of such stained basophil does not substantially reduce the intensity of light incident on photodiode 273. In the most general case, therefore, the smallest amplitude signal generated in response to the presence of a stained basophil in viewing chamber 151 of analytical channel AI is produced at the output of the photodiode 273 during a counting sequence. Therefore, rather than provide associative logic to discriminate such signals, logic channel LIII counts basophils and any noise pulses, and, also, the noise pulses individually, and subtracts such counts to provide the total basophil percentage population in the particular blood sample.

As shown, the output of photodiode 273 is connected to amplifier 275, whose output signal is connected to the input of comparator 277. Comparator 277 compares the output signal of amplifier 275, and provides an output only when the amplitude of such signal exceeds that corresponding to the presence of a stained basophil in viewing chamber 151. In effect, comparator 277 does not provide an output in response to the presence of a basophil in viewing chamber 151', rather, the output of comparator 277 is indicative of a noise signal which could be falsely counted as a basophil. The parallel comparator 207, in the basic logic arrangement of logic channel LIII, is connected to the output of photodiode 271 along amplifier 203, such comparator 207 operates, as described, to compare the output signal of amplifier 207 and provide an output when such signal exceeds a preestablished threshold. Accordingly, the output of comparator 207 in logic channel LIII is indicative of either the presence of a basophil in the viewing chamber 151 or ofa noise signal which could be falsely counted as a basophil. The outputs of comparators 207 and 277 are connected to the inputs of multivibrators 211 and 279, respectively. Accordingly, multivibrator 211 generates an output pulse in response to each basophil absorption and large noise signal, whereas the multivibrator 279' generates an output pulse only in response to a large noise signals. The outputs of multivibrators 211 and 279 are connected to one input of AND gates 213 and 281, respectively; the remaining inputs of AND gates 213 and 281 are connected to the output of multivibrator 209 in logic channel LIII, so as to be gated upon the coincidence of a scatter signal, as described. The outputs of multivibrator 209 and AND gates 213 and 281 are connected to one input of blocking AND gates 215, 217 and 283, respectively, in logic channel LIII; the remaining inputs of blocking AND gates 215, 217, and 283 are connected to the set output of flip-flop 219. When flip-flop 219 is set and blocking AND gates 215, 217, and 283 are enabled during a counting cycle, scatter pulses generated by multivibrator 209 are counted in preset counter 231, whereas accumulating counters 233 and 285 are operative to count absorption pulses generated by multivibrator 21 1 and 279, respectively, in coincidence with such scatter pulses.

The outputs of accumulation registers 233 and 285 in logic channel LIII are connected to respective inputs of a subtract register 287. When the counting cycle in logic channel LIII is completed, as indicated by a full count in preset counter 231, preset counter operates to trigger subtract register 287 along lead 237-289, so as to subtract the total counts in the accumulation registers 233 and 285, and store the difference indicative of the total number of basophils in the total leukocyte count indicated by preset register 231 of logic channel LIII. When the subtract register has completed its operation, it energizes one input of AND gate 291, whose other input has been previously energized by the preset counter 231 of logic channel LIII. The output of AND gate 291 is connected to one input of start-scan AND gate 239, which input is energized to indicate that the basophil count in logic channel LIII has been completed.

At the completion of each counting operation, there fore, the total lymphocyte count is stored in the accumulating counter 267 in logic channel LI; the total neutrophil count is stored in subtract register 247, which is responsive to the accumulating counters 233 in logic channels LI and LII, respectively; the total eosinophil count is stored in accumulating counter 233 of logic channel LII; the total basophil count is stored in the subtract register 287 responsive to accumulating counter 233 and 285 of logic channel LIII; and the total monocyte count is stored in accumulating counter 233 in logic channel LIV. The outputs of each of these registers are connected to corresponding inputs of multiplexer 241, as shown. The total percentage population of each respective cell type is given by the ratio of its corresponding count to the count indicated by its corresponding preset register 231. Advantageously, the preset counters 231 in each of the logic channels LI,

LII, LIII, and LIV are limited to a same number, preferably, 10,000, whereby the conversion to percentage population of the individual cell types is given directly by the first-two significant digits of the individual count contained in the corresponding accumulating counter, and avoids the need for additional ratioing circuitry.

Multiplexer 241 is operative, at. least, to readout he first-two significant digits from each, of the registers, preferably, on a sequential basis. When the counting sequence has been completed, each input of the startscan AND gate 239 has been energized, and a control pulse is directed along lead 293 to trigger multiplexer 241. The information from each of accumulating counter 267 in logic channel LI (lymphocyte), subtract register 247 (neutrophil), accumulating counter 233 in logic channel LII (eosinophil) subtract register 287 (basophil), and accumulating register 233 in logic channel LIV (monocyte), is directed, in turn, to printer 221. Printer 221 permanently records the percentage populations of each of the different cell types contained in a same blood sample and as stored in these registers in correlated fashion. When multiplexer 241 has completed its scan and printer 221 has completed its recording operation, printer 221 generates a control pulse, or level, along lead 235 to reset each of the flip flops 219 and concurrently reset each of preset count ers 231 and accumulating counters 233 in logic channels LI, LII, LIII, and LIV, along with the additional accumulating counters 267 and 285 in logic channels LI and LIII, respectively. Accordingly, a next control pulse directed along lead 227 indicating the presence of stained segments of a next blood sample in the quotient streams passing through viewing windows 151 of analytical channels AI, AII, AIIl, and AIV, respectively, operates to enable each of the AND gates 223 which, in turn, set flip-flop 219 in each logical channel and commence a next counting operation with respect to the next sample.

What is claimed is:

1. Apparatus fordetermining the percentage population of a selected type of particulate within a group of particulates contained in a sample comprising means for treating said sample to alter the characteristics of a selected type of particulate,

means for viewing at least a portion of said treated sample,

first means responsive to said viewing means for counting a predetermined number of particulates in said treated sample portion having either altered or unaltered characteristics to define a reference within which to count the population of said selected particulates,

second means responsive to said viewing means for discriminating said selected particulates on the basis of said altered characteristics and for counting said selected particulates in said treated sample portion within said reference, and

means for relating the respective counts of said first and second counting means for determining the percentage population of said selected particulates in said sample.

2. Apparatus as claimed in claim 1 wherein said sample is a liquid containing a suspension of particulates, and further including means for passing said treated liquid sample as a stream along a viewing chamber,

said treating means being operative to alter the spectral characteristics of said selected particulates,

said viewing means including means for directing a transverse light beam through said viewing chamher,

said first and second means being responsive to said light beam directed through said viewing chamber to count said particulates having altered or unaltered characteristics and to count said particulates having altered characteristics, respectively, while passing along said viewing chamber.

3. Apparatus as claimed in claim 2 wherein said first means includes means for detecting the passage along said viewing chamber of said particulates having altered or unaltered spectral characteristics with respect to a characteristic common to said altered and unaltered particulates, and said second means includes means for detecting the passage along said viewing chamber of said selected particulates with respect to their altered spectral characteristics.

4. Apparatus according to claim 2 wherein said first and second means are operative with respect to particulates being passed through a same portion of said viewing chamber, said first and second means being operative concurrently to count particulates having altered or unaltered characteristics and to count said particulates having altered characteristics, respectively.

5. Apparatus according to claim 4 wherein said first means is limited at a predetermined count and further including means for inhibiting said second means when said predetermined count has been attained by said first means, whereby the count attained by said second means indicates the population of said selected particulate within said predetermined count.

6. Apparatus as claimed in claim 3, wherein,

. said first and second means, include first and second generating means, respectively, to provide an indication upon the detection of a particulate passing along said viewing chamber,

first and second counter means for counting indications provided by said first and second generating means, respectively, said second counter means being operative to count indications provided by said second generating means only on the coincidence of an indication being provided to said first counter by said first generating means.

7. Apparatus according to claim 2 further including pressure means for passing said liquid through said viewing chamber, said pressure means including a high flow-resistance coil in fluid flow communication with said viewing chamber and said pressure means.

8. Apparatus as claimed in claim 1, wherein said medium is a liquid sample containing a suspension of particulates,

means for passing said liquid sample as a stream, said passing means including further means for dividing said liquid .sample into a plurality of quotient streams, said treating means being operative to treat each of said quotient streams on an individual basis for altering the characteristics of a different selected type of particulates in each of said quotient streams and while retaining the respective characteristics of other particulates in each of said quotient streams substantially unaltered, said viewing means being operative to view at least a portion of each of said treated quotient streams,

said first means being operative to detect and count particulates in each of said quotient streams having either altered or unaltered characteristics to define a reference corresponding to said each quotient stream,

said second means being operative to discriminate said selected particulates in said each of said quotient streams on the basis of said altered characteristics and to count said selected particulates in said each quotient stream within said corresponding reference, and

means for relating the respective counts of said first and second means corresponding to said each quotient stream for determining the percentage populations of said selected particulates in said liquid sample, said defined references for each of said quotient streams being related, whereby the respective percentage populations of each of said selected particulates in said lIquid sample are related. 9. An apparatus for determining the percentage population of a selected type of leukocyte in a blood sample, comprising means for treating a blood sample to selectively stain,

at least, a selected type of leukocyte therein,

means for viewing at least a portion of said treated sample to discriminate and detect stained leukocytes and, also, to detect stained and unstained leukocytes in said portion,

first means responsive to said viewing means for counting said detected stained leukocytes in said portion of said blood sample to provide a first count,

second means responsive to said viewing means for counting each of said detected stained and unstained leukocytes in portion of said blood sample to provide a second count, the ratio of said first and second counts being indicative to the percentage population of said selected type of leukocyte in said blood sample.

10. Apparatus according to claim 9, wherein said second counting means is limited at a predetermined number, and further including means responsive to said second counting means for inhibiting said first counting means when said predetermined number has been counted by said second counting means.

1 1. Apparatus according to claim 9, further including means for operating said first counting means only in concurrence with the operation of said second counting means.

12. Apparatus according to claim 9, further including means for passing said treated blood sainple as a flowing stream through a viewing chamber, and said viewing means including optical means associated with said viewing chamber for discriminating and detecting said stained leukocytes and for detecting stained and un stained leukocytes passing in said flowing stream along at least a portion of said viewing chamber, said first and second counting means being responsive to said optical means, said optical means including means for passing light transversely through said portion of said viewing chamber first means for detecting absorption of said light beam to indicate passage of said stained leukocytes along said portion of said viewing chamber; and

second means for detecting scattering of said light beam to indicate passage of stained and unstained leukocytes along said portion of said viewing chamber, said first and second counting means being responsive to-said first and second detecting means, respectively.

13. Apparatus for determining the respective percentagerpopulations of selected leukocytes in whole blood samples, comprising,

means for passing successive blood samples as a continuous stream means for dividing said continuous stream into a plurality of quotient streams, each of said quotient streams including portions of each of said successive samples,

means for treating each of said quotient streams to detectably alter the characteristics of different selected ones of said leukocytes in said quotient streams,

means for viewing at least a portion of each of said treated quotient streams,

said viewing means including first means for discriminating and counting selected leukocytes on the basis of said altered characteristics in each of said sample portions contained in said quotient streams, so as to provide a first count for each sample portion,

said viewing means further including second means for detecting and counting leukocytes having altered and unaltered characteristics in each of said sample portions contained in said quotient streams, so as to provide a second count for each sample portion, the ratio of said first and second counts corresponding to a sample portion in said quotient streams indicating the respective percentage population of said selected leukocytes in the original blood, and

means for correlating the respective percentage populations of said selected leukocytes in said corresponding portions of said original blood sample.

14. Apparatus according to claim 13, further including means for separating said portions of each of said successive samples along said quotient streams by an inert fluid segment.

15. Apparatus according to claim 13, wherein said treating means includes means for detectably altering the characteristics of leukocytes in a selected class of leukocytes in at least one of said quotient streams.

16. Apparatus according to claim 15, wherein said first means are operative to discriminate and count individual leukocytes in said selected class in said one quotient stream on the basis of said altered characteristics, so as to provide a third count, the ratio of said third count and said second count provided by said second means indicating the percentage population of said selected class of leukocytes in said original blood sample.

17. Apparatus according to claim 16, wherein particular ones of said leukocytes in said selected class have been treated in another of said quotient streams, and further including means for substracting the percentage population of said particular leukocytes from the percentage population of said selected class of leukocytes, so as to indicate the percentage population in said original blood sample of remaining leukocytes in said selected class.

18. Apparatus according to claim 13, wherein said second means, as associated with at least one of said quotient streams, includes means for discriminating and counting particular leukocytes in said one stream on the basis of an unaltered characteristic of said particular leukocytes.

19. Apparatus according to claim 17, wherein said treating means are operative to stain so as to change the spectral characteristics of a class of leukocytes along at least one quotient stream, while leaving remaining leukocytes in said one quotient stream substantially unchanged, and further including means for passing said one quotient stream along a viewing chamber having an unobstructed light passageway,

means for passing light along said passageway,

said first means being responsive to an absorption of said light beam by each of said leukocytes having changed spectral characteristics and said second means being responsive to a scattering of light by said stained and unstained leukocytes, the scattering of light being a direct function of the cellular size of a leukocyte, and

third means responsive to said second means for discriminating and identifying particular unstained ones of said leukocytes in said class according to the magnitude of the scattered light signal.

20. Apparatus according to claim 19, further including means responsive to said third means for counting said particular unstained leukocytes, the ratio of the respective counts of said third means and said second means associated with said one quotient stream being indicative of the percentage population of said particular unstained leukocyte in said original blood sample.

21. Apparatus according to claim 20, further including gating means connecting said second means responsive to a scattering of light and said third means for counting said particular unstained leukocytes, said gating means being inhibitive by said first means during detection of a stained leukocyte by said first means. 

1. Apparatus for determining the percentage population of a selected type of particulate within a group of particulates contained in a sample comprising means for treating said sample to alter the characteristics of a selected type of particulate, means for viewing at least a portion of said treated sample, first means responsive to said viewing means for counting a predetermined number of particulates in said treated sample portion having either altered or unaltered characteristics to define a reference within which to count the population of said selected particulates, second means responsive to said viewing means for discriminating said selected particulates on the basis of said altered characteristics and for counting said selected particulates in said treated sample portion within said reference, and means for relating the respective counts of said first and second counting means for determining the percentage population of said selected particulates in said sample.
 2. Apparatus as claimed in claim 1 wherein said sample is a liquid containing a suspension of particulates, and further including means for passing said treated liquid sample as a stream along a viewing chamber, said treating means being operative to alter the spectral characteristics of said selected particulates, said viewing means including means for directing a transverse light beam through said viewing chamber, said first and second means being responsive to said light beam directed through said viewing chamber to count said particulates having altered or unaltered characteristics and to count said particulates having altered characteristics, respectively, while passing along said viewing chamber.
 3. Apparatus as claimed in claim 2 wherein said first means includes means for detecting the passage along said viewing chamber of said particulates having altered or unaltered spectral characteristics with respect to a characteristic common to said altered and unaltered particulates, and said second means includes means for detecting the passage along said viewing chamber of said selected particulates with respect to their altered spectral charactEristics.
 4. Apparatus according to claim 2 wherein said first and second means are operative with respect to particulates being passed through a same portion of said viewing chamber, said first and second means being operative concurrently to count particulates having altered or unaltered characteristics and to count said particulates having altered characteristics, respectively.
 5. Apparatus according to claim 4 wherein said first means is limited at a predetermined count and further including means for inhibiting said second means when said predetermined count has been attained by said first means, whereby the count attained by said second means indicates the population of said selected particulate within said predetermined count.
 6. Apparatus as claimed in claim 3, wherein, said first and second means, include first and second generating means, respectively, to provide an indication upon the detection of a particulate passing along said viewing chamber, first and second counter means for counting indications provided by said first and second generating means, respectively, said second counter means being operative to count indications provided by said second generating means only on the coincidence of an indication being provided to said first counter by said first generating means.
 7. Apparatus according to claim 2 further including pressure means for passing said liquid through said viewing chamber, said pressure means including a high flow-resistance coil in fluid flow communication with said viewing chamber and said pressure means.
 8. Apparatus as claimed in claim 1, wherein said medium is a liquid sample containing a suspension of particulates, means for passing said liquid sample as a stream, said passing means including further means for dividing said liquid sample into a plurality of quotient streams, said treating means being operative to treat each of said quotient streams on an individual basis for altering the characteristics of a different selected type of particulates in each of said quotient streams and while retaining the respective characteristics of other particulates in each of said quotient streams substantially unaltered, said viewing means being operative to view at least a portion of each of said treated quotient streams, said first means being operative to detect and count particulates in each of said quotient streams having either altered or unaltered characteristics to define a reference corresponding to said each quotient stream, said second means being operative to discriminate said selected particulates in said each of said quotient streams on the basis of said altered characteristics and to count said selected particulates in said each quotient stream within said corresponding reference, and means for relating the respective counts of said first and second means corresponding to said each quotient stream for determining the percentage populations of said selected particulates in said liquid sample, said defined references for each of said quotient streams being related, whereby the respective percentage populations of each of said selected particulates in said lIquid sample are related.
 9. An apparatus for determining the percentage population of a selected type of leukocyte in a blood sample, comprising means for treating a blood sample to selectively stain, at least, a selected type of leukocyte therein, means for viewing at least a portion of said treated sample to discriminate and detect stained leukocytes and, also, to detect stained and unstained leukocytes in said portion, first means responsive to said viewing means for counting said detected stained leukocytes in said portion of said blood sample to provide a first count, second means responsive to said viewing means for counting each of said detected stained and unstained leukocytes in portion of said blood sample to provide a second count, the ratio of said first and second counts being indicative to the percentAge population of said selected type of leukocyte in said blood sample.
 10. Apparatus according to claim 9, wherein said second counting means is limited at a predetermined number, and further including means responsive to said second counting means for inhibiting said first counting means when said predetermined number has been counted by said second counting means.
 11. Apparatus according to claim 9, further including means for operating said first counting means only in concurrence with the operation of said second counting means.
 12. Apparatus according to claim 9, further including means for passing said treated blood sample as a flowing stream through a viewing chamber, and said viewing means including optical means associated with said viewing chamber for discriminating and detecting said stained leukocytes and for detecting stained and unstained leukocytes passing in said flowing stream along at least a portion of said viewing chamber, said first and second counting means being responsive to said optical means, said optical means including means for passing light transversely through said portion of said viewing chamber ; first means for detecting absorption of said light beam to indicate passage of said stained leukocytes along said portion of said viewing chamber; and second means for detecting scattering of said light beam to indicate passage of stained and unstained leukocytes along said portion of said viewing chamber, said first and second counting means being responsive to said first and second detecting means, respectively.
 13. Apparatus for determining the respective percentage populations of selected leukocytes in whole blood samples, comprising, means for passing successive blood samples as a continuous stream means for dividing said continuous stream into a plurality of quotient streams, each of said quotient streams including portions of each of said successive samples, means for treating each of said quotient streams to detectably alter the characteristics of different selected ones of said leukocytes in said quotient streams, means for viewing at least a portion of each of said treated quotient streams, said viewing means including first means for discriminating and counting selected leukocytes on the basis of said altered characteristics in each of said sample portions contained in said quotient streams, so as to provide a first count for each sample portion, said viewing means further including second means for detecting and counting leukocytes having altered and unaltered characteristics in each of said sample portions contained in said quotient streams, so as to provide a second count for each sample portion, the ratio of said first and second counts corresponding to a sample portion in said quotient streams indicating the respective percentage population of said selected leukocytes in the original blood, and means for correlating the respective percentage populations of said selected leukocytes in said corresponding portions of said original blood sample.
 14. Apparatus according to claim 13, further including means for separating said portions of each of said successive samples along said quotient streams by an inert fluid segment.
 15. Apparatus according to claim 13, wherein said treating means includes means for detectably altering the characteristics of leukocytes in a selected class of leukocytes in at least one of said quotient streams.
 16. Apparatus according to claim 15, wherein said first means are operative to discriminate and count individual leukocytes in said selected class in said one quotient stream on the basis of said altered characteristics, so as to provide a third count, the ratio of said third count and said second count provided by said second means indicating the percentage population of said selected class of leukocytes in said original blood sample.
 17. Apparatus according to claim 16, wherein particular ones of said leukocytes in said selected class have been treatEd in another of said quotient streams, and further including means for substracting the percentage population of said particular leukocytes from the percentage population of said selected class of leukocytes, so as to indicate the percentage population in said original blood sample of remaining leukocytes in said selected class.
 18. Apparatus according to claim 13, wherein said second means, as associated with at least one of said quotient streams, includes means for discriminating and counting particular leukocytes in said one stream on the basis of an unaltered characteristic of said particular leukocytes.
 19. Apparatus according to claim 17, wherein said treating means are operative to stain so as to change the spectral characteristics of a class of leukocytes along at least one quotient stream, while leaving remaining leukocytes in said one quotient stream substantially unchanged, and further including means for passing said one quotient stream along a viewing chamber having an unobstructed light passageway, means for passing light along said passageway, said first means being responsive to an absorption of said light beam by each of said leukocytes having changed spectral characteristics and said second means being responsive to a scattering of light by said stained and unstained leukocytes, the scattering of light being a direct function of the cellular size of a leukocyte, and third means responsive to said second means for discriminating and identifying particular unstained ones of said leukocytes in said class according to the magnitude of the scattered light signal.
 20. Apparatus according to claim 19, further including means responsive to said third means for counting said particular unstained leukocytes, the ratio of the respective counts of said third means and said second means associated with said one quotient stream being indicative of the percentage population of said particular unstained leukocyte in said original blood sample.
 21. Apparatus according to claim 20, further including gating means connecting said second means responsive to a scattering of light and said third means for counting said particular unstained leukocytes, said gating means being inhibitive by said first means during detection of a stained leukocyte by said first means. 